Metabolomics combined with network pharmacology reveals a role for astragaloside IV in inhibiting enterovirus 71 replication via PI3K-AKT signaling

Background Astragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms. Methods The GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID50), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman’s algorithm. Results This study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis. Conclusion This study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12967-024-05355-9.


Introduction
Hand-foot-mouth disease (HFMD) is caused by a multitude of enteroviral infections and poses a significant public health threat to children [1].Enterovirus 71 (EV71), a common pathogen of HFMD, is classified as an enterovirus in the family of small RNA viruses [2].EV71 commonly infects children under the age of 5 years, and the resulting HFMD is typically self-limiting.However, a small proportion of severely ill patients can develop pulmonary edema, myocarditis, and potentially fatal encephalitis in newborns [3].However, many clinical investigations have shown that HFMD can be mitigated by diet or dietary components.
Viral infections often disrupt the body's oxidative stress response, leading to compromised immune function, apoptosis, inflammation, and organ and tissue dysfunction that contribute to patient pathogenesis.HBV and HIV infection induces oxidative stress damage by producing reactive oxygen species (ROS) in infected cells [4,5], while the excessive production of ROS can induce apoptosis (Giorgio et al., 2005).Nuclear Factor E2-Related Factor 2 (Nrf2) serves a crucial role in the antioxidant stress response as a regulatory protein downstream of ROS [6].Nrf2 activators [7] or the activation of Nrf2 [8,9] can exhibit antiviral effects.The PI3K/ AKT signaling pathway is implicated in the regulation of apoptosis, cell proliferation, and viral infection [10][11][12].The use of PI3K/AKT inhibitors has been shown to promote infection by infectious spleen and kidney necrosis viruses [13], indicating that PI3K/AKT could be a potential therapeutic target for viral diseases.
Astragalus membranaceus (Fisch.)Bunge, is commonly used in traditional formulas to prevent the invasion of external pathogens by strengthening the lungs and spleen [14], treat human immunodeficiency virus (HIV) infection [15], suppress SARS-CoV-2 entry [16], protect acute kidney injury [17].Astragaloside IV (AST-IV, chemical formula: C41H68O14, molecular weight: 785), one of the active ingredients of Astragalus membranaceus (Fisch.)Bunge, has the pharmacological effects of anti-inflammatory, immunomodulatory, antibacterial and antiviral properties [18,19].Recent studies have showed that AST-IV inhibits the replication of dengue virus [20], hepatitis B virus [21], adenovirus [22].It has also been found to reduce coxsackievirus B3-induced apoptosis in cardiomyocytes [23].However, the inhibitory effect of AST-IV on EV71 virus replication and its underlying mechanism are not yet fully understood.
Network pharmacology is a method for effectively predicting relevant targets and pathways for drug treatment of diseases to further elucidate the underlying mechanisms [24].Metabolomics has gained significant traction in disease diagnosis, drug mechanism of action studies, and the discovery of novel biomarkers [25].Therefore, we employed an in vitro model of EV71 infection and AST-IV treatment to investigate the anti-EV71 mechanism of AST-IV.Here, we found that AST-IV inhibited EV71 replication through the ROS-PI3K-AKT pathway.

Cell viability assay
GES-1 cells or RD cells were cultured in 96-well plates (1 × 10 4 /well).Subsequently, the GES-1 cells or RD cells were infected with EV71, treated with AST-IV, or treated with EV71 and AST-IV.GES-1 or RD cell viability was determined using a Cell Counting Kit-8 (CCK-8) kit (C0038, Beyotime, Shanghai, China) according to the manufacturer's protocol.

PI staining
4 µM propyl iodide (PI, ST511, Beyotime, Shanghai, China) was added to 125 µL of fresh DMEM and cocultured with the GES-1 cells in an incubator for 30 min.Then, 1 µL of Hoechst 33,342 (C1027, Beyotime, Shanghai, China) was added to 100 µL of fresh DMEM and cocultured with the GES-1 cells in an incubator for 10 min.The cells were then photographed using an inverted fluorescence microscope (Nikon, Japan).

LC-MS/MS method for GES-1 cell metabolomics
After slowly thawing the treated cells (approximately 10 ^7) at 4 °C, 1000 µL of the extraction solution (MeOH: ACN: H2O, 2:2:1 (v/v)) was added to a centrifuge tube and mixed by vortexing for 30 s. Subsequently, the GES-1 cells were frozen in liquid nitrogen, thawed at room temperature, and vortexed for 30 s and this process was repeated three times.The cells were then sonicated for 10 min on ice and kept at -40 ℃ for 1 h.The cell lysate supernatant was collected in a fresh glass vial for analysis.The supernatant was stored in a 4 °C autosampler, and 2 µL of each sample was analysed.MS/MS spectra were acquired using an Orbitrap Exploris 120 mass spectrometer controlled by Xcalibur software (Xcalibur, Thermo).

Measurements of intracellular ROS
GES-1 cells or RD cells were incubated in DMEM containing 10 µM DCFH-DA without FBS (S0033S, Beyotime, Shanghai, China) for 30 min in an incubator.After washing with PBS, the GES-1 cells or RD cells were photographed using an inverted fluorescence microscope (Nikon, Japan).The green fluorescence intensity was measured using ImageJ software.

Network pharmacology analysis
Potential targets of AST-IV were collected from the Swis-sTargetPrediction (http://www.swisstargetprediction. ch/) and TargetNet (http://targetnet.scbdd.com/)databases.Protein names were converted to gene names in the UniProts database.We entered the keyword " hand foot mouth disease " in the GeneCards (https://www.genecards.org/),DisGenet (https://www.disgenet.org/),and OMIM (https://www.omim.org/)databases to identify HFMD-related therapeutic targets.We also performed a collection of RNA-sequencing (GSE15323) from the Gene Expression Omnibus (GEO) database following EV71 infection and the DEGs were screened by Gene Expression Omnibus (GEO) 2R analysis with an adjustable P < 0.05 and a |log2-fold change (FC)| > 1.0.The HFMD targets and AST-IV targets were entered into the online platform (https://www.bioinformatics.com.cn/), and the common targets were identified using a Venn diagram.Protein-protein interaction (PPI) networks of common genes were constructed using the STRING database (https://cn.string-db.org/) with interaction scores > 0.4.Cytoscape 3.9.0 was used to visualize the PPI networks.Fill colors, heights, and widths were mapped continuously by degree.PPI network construction of key genes was performed and visualized using Metascape (https://metascape.org/).Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed and visualized using an online platform (https://www.bioinformatics.com.cn/,https://metascape.org/).Hub genes were selected by the Cytoscape 3.9.0plugin cyto-Hubba [28], and the most critical hub genes were identified by the following algorithms: closeness, MCC, degree, MNC, radiality, stress, and EPC.

Molecular docking
The crystal structures of the AKT1 (PDB ID: 1h10) and PIK3R1 (PDB ID: 1pbw) proteins were downloaded from the Protein Data Bank (PDB, https://www.rcsb)database, and the structure of AST-IV was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/).Docking validation was performed using AutoDock 1.5.6 software.During the molecular docking process involving AKT1, PIK3R1 and AST-IV, a grid was created with dimensions x = 56.3333333333,y = 52.0,z = 65.0 or x = 98.0, y = 100.0,z = 116.0respectively, and a number of steps set to 10.The grid was defined to encompass the proteins for blind docking.Autodock generated 9 complexes as a result of the docking simulation.All of the above docking is semi-flexible.The docking results were visualized using PyMOL software.

Statistical analysis
All the data are presented as the mean ± standard deviation (SD).Statistical analysis was performed using GraphPad Prism 8.0.2 software (CA, USA).Comparisons between two groups or multiple groups were assessed using unpaired Student's t-test or one-way ANOVA with Tukey's post hoc test, respectively.Data with unequal variances were analyzed by Welch's test with the Games-Howell post hoc test.p < 0.05 was considered to indicate statistical significance.Differential metabolites were identified using a combination of the variable importance in projection (VIP) value from the OPLS-DA model and P values from Student's t test on normalized peak areas.VIP ≥ 1 and p < 0.05were considered significantly altered metabolites.

AST-IV inhibits EV71 replication
To investigate the effect of AST-IV on EV71 replication, GES-1 cells were treated with AST-IV.AST-IV at 0-10 µg/mL had no significant effect on cell viability (Fig. 1B).Next, GES-1 cells or RD cells were pretreated with AST-IV for 2 h and then infected with EV71 for 24 h-12 h.Cell viability was determined by the CCK8 assay, viral structural protein VP1 levels were determined by Western blotting, and viral titers in the supernatants were assayed through the TCID 50 method.The results showed that AST-IV increased the viability of EV71infected GES-1 cells or RD cells (Fig. 1C, Fig S1).In addition, the levels of VP1 protein (Fig. 1D, E) and viral titers in the supernatant (Fig. 1F) were reduced in AST-IV-or RBV-treated and EV71-infected GES-1 cells, indicating a decrease in viral replication.Based on these results, it can be concluded that AST-IV effectively inhibited the EV71 replication.

Effects of AST-IV on EV71-induced apoptosis
Oxidative stress can activate apoptosis, and inhibition of oxidative stress protects cells from cytotoxicity and apoptosis [29,30].To investigate the protective effects of AST-IV on EV71-induced apoptosis, we performed PI staining to assess cell viability and Western blot to measure cleaved PARP and cleaved caspase-3 levels.The results indicated that EV71 infection significantly increased the number of PI-positive cells.However, AST-IV treatment effectively reduced it, suggesting that AST-IV has a protective effect against EV71-induced cytotoxicity (Fig. 2A, B).Furthermore, we found that cleaved PARP and cleaved caspase-3 levels were significantly increased in EV71-infected GES-1 cells, indicating the activation of apoptosis.However, AST-IV inhibited the degradation of the PARP and caspase-3 proteins induced by EV71 infection, suggesting that AST-IV can protect against EV71-induced apoptosis (Fig. 2C-E).Our results demonstrated that AST-IV protects against EV71induced apoptosis in GES-1 cells.

Analysis of the metabolic profile of EV71-infected and AST-IV-treated GES-1 cells
To better understand how AST-IV exerts its anti-EV71 effects by modulating metabolites, we investigated the metabolomics of GES-1 cells.We observed an apparent separation in the OPLS-DA (Fig. 3A, C, E) and PCA (Fig. 3B, D, F) score plots among the Control, EV71, AST-IV and EV71 + AST-IV groups.The metabolic profiles of GES-1 cells were significantly different among the four groups.We used volcano and Venn diagrams to show the intrinsic connections of different metabolites.There were 293 metabolites with significant differences between the control group and the EV71 group (Fig. 4A), 196 metabolites between the control group and the AST-IV group (Fig. 4B), and 302 metabolites between the EV71 group and the AST-IV and EV71 group (Fig. 4C).Notably, we identified a total of 114 common differential metabolites among the four groups (Fig. 4D).Among these metabolites, eight were significantly altered in GES-1 cells treated with AST-IV alone.These metabolites included hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, oxypurinol, 6-hydroxy-1 H-indole-3-acetamid, and PC (14:0/15:0) (Fig. 5A-H).Furthermore, the metabolite PC (14:0/15:0) decreased (Fig. 5H), whereas hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, oxypurinol, and 6-hydroxy-1 H-indole-3-acetamid (Fig. 5A-G) increased in the AST-IV group compared to the control group.Furthermore, AST-IV treatment of EV71-infected cells restored the levels of these metabolites to essentially the same levels as those in the control group.Specifically, AST-IV increased the levels of hypoxanthine, adenine, nicotinic acid mononucleotides, and oxypurinol, indicating its effect on nucleotide metabolism.AST-IV also increased the levels of 2-ketobutyric acid, indicating its effect on cellular energy production and the citric acid cycle.In addition, AST-IV upregulated 6-hydroxy-1 H-indol-3-acetamide, suggesting its influence on cellular growth hormone metabolism.AST-IV was found to upregulate prostaglandin H2, indicating its involvement in fatty acid metabolism.Furthermore, AST-IV downregulated PC (14:0/15:0), suggesting its impact on phospholipid metabolism.Taken together,

Effects of AST-IV on antioxidative activity
Studies have shown a strong relationship between oxidative stress and viral replication [31,32].Therefore, we assayed the ROS levels and oxidative stress biomarkers in different treatment groups and the results showed that AST-IV treatment reduced ROS levels and increased the activity of CAT, GSH-Px, and T-AOC while decreasing MDA levels in EV71-infected GES-1 cells or RD cells (Fig. 6A-G, Fig S2A-F).These findings indicate that AST-IV can effectively protect GES-1 cells and RD cells from EV71-induced oxidative damage.As an ROS inhibitor, we investigated the anti-EV71 effects of NAC.Our results showed that NAC treatment reduced VP1 protein levels and viral titers in the supernatants of EV71-infected GES-1 cells (Fig. 6H-J), similar to the effects of AST-IV.This finding suggested that both AST-IV and NAC may inhibit EV71 replication by reducing oxidative stress.We concluded that AST-IV exerts anti-EV71 effects by inhibiting EV71-induced oxidative stress.

Targets of AST-IV and HFMD
To further investigate the mechanism of AST-IV in the treatment of HFMD at the protein level, we conducted network pharmacology analysis.Targets for HFMD were collected from 3 databases: 535 targets from the OMIM database, 63 targets from the DisGenet database, and 1470 targets from the GeneCards database.In addition, we included an RNA sequence (GSE157282) from the GEO database and used the criteria of |log 2 FC| > 1 and adjustable P < 0.05 to identify DEGs.By integrating the targets in the database and the DEGs from GSE157282, a total of 2223 therapeutic targets related to HFMD were identified (Fig. 7A).The AST-IV targets were collected from two databases, 100 from the Swiss TargetPrediction database and 67 from the TargetNet database, and after removing duplicate targets, a total of 100 AST-IV targets were collected (Fig. 7B).The Venn diagram showed 44 common targets between the AST-IV and HFMD (Fig. 7D).The targets were visualized as an AST-IV-target network (Fig. 7C) and an AST-IV-common targets-HFMD network (Fig. 7E) using Cytoscape 3.9.0software.AST-IV is shown in green, HFMD in orange and the targets in yellow.

Network construction and analysis
We also constructed a PPI network of 44 common targets by using Cytoscape 3.9.0 to explore the interactions between these potential targets and screened the top 10 ranked hub genes by degree value, which were AKT1, EGFR, SRC, STAT3, HSP90AA1, IL2, MDM2, NR3C1, MTOR, and AR (Fig. 8A).We also screened the top 10 ranked hub genes via the Cytoscape 3.9.0cytoHubba plugin, identified 6 common hub genes, namely, AKT1, SRC, STAT3, EGFR, HSP90AA1, and MDM2 via a Venn diagram (Fig. 8B), and constructed a PPI network via Cytoscape 3.9.0 (Fig. 8C).We also performed GO and KEGG enrichment analyses on 44 common targets.GO enrichment revealed that the inhibition of EV71 replication by AST-IV was associated with the response to drugs, thecellular response to external stimuli, the regulation of lipid metabolic process, regulation of reactive oxygen species biosynthetic process, cellular response to oxidative stress, regulation of purine nucleotide metabolic process, activation of protein kinase B activity, regulation of purine nucleotide biosynthetic process, apical plasma membrane (Fig. 8D).KEGG enrichment analysis indicated that the hub genes were mainly enriched in PI3K-AKT signaling pathway, cAMP signaling pathway, and lipid and atherosclerosis signaling pathway (Fig. 8E).To further identify more important target proteins and pathways, we performed KEGG analysis using Metascape (https://Metascape.org/),and key targets were closely related to the PI3K-AKT pathway (Fig. 8F).

The effects of AST-IV on the PI3K-AKT pathway in EV71infected and AST-IV-treated GES-1 cells
The PI3K-AKT pathway is involved in the regulation of cellular processes such as apoptosis [33] and viral replication [13].The effects of AST-IV on the PI3K-AKT pathway were investigated in EV71-infected and AST-IVtreated GES-1 cells.First, GES-1 cells were infected with EV71 at different MOIs (1, 3, and 5) for 24 h.The protein levels of p-PI3K and p-AKT were assessed by Western blot.We observed that the levels of p-PI3K and p-AKT were reduced in a dose-dependent manner in the EV71infected group compared to those in the control group (Fig. 9A-C).This finding suggested that EV71 infection inhibits the activation of the PI3K-AKT pathway in GES-1 cells.Next, GES-1 cells were treated with AST-IV for 24 h, and the protein levels of p-PI3K, t-PI3K, p-AKT, and t-AKT were detected by Western blot.The results showed that p-PI3K and p-AKT levels were increased in AST-IV-treated GES-1 cells, suggesting that AST-IV activated the PI3K-AKT pathway (Fig. 9D-F).Furthermore, we observed that AST-IV treatment reversed the inhibition of the PI3K-AKT signaling pathway caused by EV71 infection (Fig. 9G-I).This suggests that AST-IV has the ability to counteract the negative effects of EV71 on the PI3K-AKT pathway in GES-1 cells.To further validate the Western blot results, we used a molecular docking method to predict the interactions between AST-IV and AKT1 or PIK3R1.The docked binding energies between AST-IV and the AKT1 and PIK3R1 proteins were − 7.12 ± 0.35 and − 7.47 ± 0.25 kcal/mol, respectively (Fig. 9J, K, S3).This shows that there is good bonding between them (binding energy ≤-5 kcal/mol).In conclusion, our findings indicate that AST-IV treatment activates the PI3K-AKT pathway and reverses its inhibition by EV71 infection in GES-1 cells.

Discussion
We observed that medium and high concentrations of AST-IV intervention in EV71-infected GES-1 cells led to a reduction in VP1 protein levels and viral titers in the supernatant.This finding suggested that AST-IV inhibits EV71 replication.We investigated the mechanism by which AST-IV inhibits EV71 replication via network pharmacology and metabolomics analyses.Network pharmacology indicated that AT-IV may inhibit EV71 replication by AKT1, HSP90 and EGFR et al. core targets to regulate lipid metabolic processes, the cellular response to oxidative stress, purine nucleotide metabolic processes, the PI3K-AKT signaling pathway, the cAMP signaling pathway, and lipid and atherosclerosis signaling pathways.The AKT protein has been shown to be activated in the early stages of EV71 infection [34], and inhibiting the expression of the HSP90 [35] and EGFR [36] proteins can inhibit EV71 replication.Metabolite profiling of the control, EV71, AST-IV, and EV71 and AST-IV groups revealed significant changes in eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, oxypurinol, 6-hydroxy-1 H-indole-3-acetamide and PC (14:0/15:0).Importantly, the levels of all eight metabolites were restored to normal cellular levels when AST-IV was applied to EV71-infected cells.
ROS, whether exogenously or endogenously overproduced, can lead to an imbalance in redox homeostasis [42].Although elevated levels of virus-induced ROS  trigger innate antiviral immunity, a recent study reported that ROS production is beneficial for viral replication [43].This phenomenonhas been observed for alphaviruses and flaviviruses, which exhibit enhanced replication in an oxidized cellular environment [44].In addition, elevated levels of ROS have been detected in SARS-CoV-2-infected patients, and early diagnosis of COVID-19 can be achieved through real-time detection of ROS levels in fresh sputum by electrochemical tracing [45].Nrf2, an antioxidant gene, inhibits the replication of vesicular stomatitis virus (VSV) and influenza A virus (H1N1) by suppressing virus-induced oxidative stress [8,46].In the context of EV71 infection, the virus was found to induce mitochondrial oxidative stress to promote its own replication [47].In our study, we observed that EV71 infection caused an increase in cellular ROS production.However, intervention with AST-IV reduced the ROS levels caused by EV71 infection, and the ROS inhibitor NAC inhibited EV71 replication.Moreover, AST-IV treatment of EV71-infected GES-1 cells or RD cells restored the levels of oxidative stress-related markers such as CAT, GSH-Px, T-AOC, and MAD to normal levels.These findings suggest that AST-IV may inhibit EV71 replication by upregulating the levels of oxypurinol, NAMN, and 6-Hydroxy-1 H-indole-3-acetamide to activate the body's antioxidant system.In summary, AST-IV appears to exert antiviral effects against EV71 infection by reducing ROS production and restoring the antioxidant system.
Hypoxanthine, a nucleotide produced from purines, is an important intermediate in the anabolism of purine nucleotides.The novel C-2 modified hypoxanthine has excellent anti-RNA viral activity [48].PC (14:0/15:0) is an important component of the bilayer membrane structure of the cell membrane.Viruses can use the cell membrane as a vehicle for noncleavage transport out of the cell and spread to other cells or susceptible hosts to facilitate the spread of infection [49].The levels of 2-ketobutyric acid are elevated after HIV infection and return to normal after treatment, suggesting that 2-ketobutyric acid plays an important role in the pathogenesis of HIV [50].In our study, we found that EV71 infection downregulated 2-ketobutyric acid and hypoxanthine levels and upregulated PC (14:0/15:0) levels.All of these metabolites returned to normal cellular levels after AST-IV intervention, suggesting that AST-IV may inhibit EV71 replication and spread by modulating these metabolites.
Prostaglandin E (2) (PEG2) is converted from prostaglandin H2, which can inhibit HIV replication by activating PKA and cAMP [51].Adenine also activates cAMP [52].Apoptosis is a common response to viral infection [53][54][55].Research suggests that viruses tend to exploit host cells to induce apoptosis in tissues or immune cells, allowing the virus to persist during infection [56].The EV71 [57] and IAV [58] viruses induce apoptosis in infected cells, allowing the release of daughter viruses to infect neighboring cells.In this study, AST-IV was found to inhibit EV71-induced apoptosis.The PI3K-AKT pathway is a key signaling pathway involved in the regulation of viral infection [12] and apoptosis [59].Research has shown that inhibition of the PI3K-AKT pathway can promote mammalian reovirus replication [60], while upregulation of the PI3K-AKT pathway can suppress endoplasmic reticulum stress-induced cell apoptosis [61].Both cAMP and ROS can regulate the PI3K-AKT pathway, with cAMP activating it [62] and ROS inhibiting it [63].Therefore, we focused on the PI3K-AKT pathway as the key pathway through which AST-IV exerts its anti-EV71 effect.The PI3K-AKT pathway is activated during the early stages of EV71 infection but inhibited after 1 h of infection [64].In our study, the PI3K-AKT pathway was inhibited in EV71-infected GES-1 cells for 24 h, whereas AST-IV treatment restored its activity.This study indicates that AST-IV activates the cAMP pathway and the body's antioxidant system by modulating these metabolites, which further activates the PI3K-AKT pathway, thereby inhibiting EV71-induced cell.
apoptosis and replication of EV71.In our future studies, we will first experimentally determine the targeted binding of AST-IV to PI3K and AKT.Second, we aim to validate these findings in animal models and potentially in clinical trials to assess the translational potential of AST-IV in the treatment of EV71 infection.

Conclusion
In conclusion, our research revealed that AST-IV can effectively inhibit EV71 replication.We propose that AST-IV may exert its anti-EV71 effects by targeting eight metabolites.These metabolites are likely to activate the body's antioxidant system, which further activates the PI3K-AKT pathway to inhibit EV71-induced cell apoptosis and EV71 replication.

Fig. 1
Fig. 1 AST-IV inhibits EV71 replication.(A) Chemical structure of AST-IV.(B, C) A CCK-8 assay was performed to measure the viability of GES-1 cells in different treatment groups.(D, E) Western blotting was used to analyze the levels of the viral structural protein VP1 in the different treatment groups.(F) The viral titers in the supernatants of different treatment groups were determined by TCID50 assay.The data are presented as the mean ± SD (n = 3) of at least three independent experiments.****P < 0.0001 compared with the control group; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared with the EV71infected group

Fig. 2
Fig. 2 Effects of AST-IV on EV71-induced apoptosis.(A, B) Fluorescence images of EV71-infected GES-1 cells were obtained in the presence of AST-IV for 2 h-prior.The cells were stained with PI. (C-E) Western blot analysis of the apoptosis marker proteins cleaved PARP and cleaved caspase-3 in EV71-infected GES-1 cells in the presence of AST-IV.The data are presented as the mean ± SD (n = 3) of at least three independent experiments.****P < 0.0001 compared to thecontrol group; ##P < 0.01, ###P < 0.001, ####P < 0.01 compared to the EV71-infected group

Fig. 3
Fig. 3 OPLS-DA and PCA analysis of LC-MS/MS of GES-1 cells.(A) OPLS-DA and (B) PCA score plots of the control group and EV71 group.(C) OPLS-DA and (D) PCA score plots of the control group and AST-IV group.(E) OPLS-DA and (F) PCA score plots of the EV71 group and EV71 + AST-IV group

Fig. 4
Fig. 4 Analysis of differential metabolites in the four groups.(A) The volcano plot shows the differential metabolite levels between the control group and the EV71 group.(B) The volcano plot shows the differential metabolite levels between the control group and the AST-IV group.(C) The volcano plot shows the differential metabolite levels between the EV71 group and the EV71 + AST-IV group.(D) Venn diagram showing the overlapping metabolites from the control group, EV71 group, AST-IV group and EV71 + AST-IV group

Fig. 7 Fig. 8
Fig. 7 Targets of AST-IV and HFMD.(A) Venn diagram of HFMD-related targets.(B) Venn diagram of AST-IV-related targets.(C) Venn diagram of the common targets between AST-IV and HFMD.(D) Visualization of the AST-IV-target network.(E) Visualization of the AST-IV-common targets-HFMD network

Fig. 9
Fig. 9 The effects of AST-IV on the PI3K-AKT pathway in EV71-infected and AST-IV-treated GES-1 cells.(A-C) Western blot analysis of p-PI3K and p-AKT levels in EV71-infected GES-1 cells.(D-F) The protein levels of p-PI3K, t-PI3K, p-AKT, and t-AKT in AST-IV-treated GES-1 cells were analyzed by western blot.(G-I) The protein levels of p-PI3K and p-AKT in EV71-infected and AST-IV-treated GES-1 cells were analyzed by Western blot.(J, K) The conformation of the molecular docking between AST-IV and AKT1 or PIK3R1.The data are presented as the mean ± SD (n = 3) of at least three independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group; #P < 0.05, ##P < 0.01 compared with the EV71-infected group

Fig. 10
Fig. 10 Correlation between targeted metabolites and apparent indicators.(A) Association analysis of metabolites with apparent indicators by Spearman's method.Differences were considered statistically significant at P < 0.05.(B) Network (only correlations with |R| > 0.6 with P < 0.05 were presented) among metabolites and apparent indicators.Red nodes represent differential metabolites, and purple nodes represent apparent indicators.Red lines indicate positive correlations, while purple lines indicate negative correlations